Spatial and temporal regulation of Wnt signaling pathway members in the development of butterfly wing patterns

Wnt signaling members are involved in the differentiation of cells associated with eyespot and band color patterns on the wings of butterflies, but the identity and spatio-temporal regulation of specific Wnt pathway members remains unclear. Here, we explore the localization and function of Armadillo/β-catenin dependent (canonical) and Armadillo/β-catenin independent (noncanonical) Wnt signaling in eyespot and band development in Bicyclus anynana by localizing Armadillo (Arm), the expression of all eight Wnt ligand and four frizzled receptor transcripts present in the genome of this species and testing the function of some of the ligands and receptors using CRISPR-Cas9. We show that distinct Wnt signaling pathways are essential for eyespot and band patterning in butterflies and are likely interacting to control their active domains.


INTRODUCTION
Wnt signaling is fundamental to cellular communication in multicellular organisms. This communication involves secreted glycoproteins, the Wnt ligands, that are produced in signaling cells, traveling some distance away to regulate the expression of target genes in surrounding cells (1,2). Understanding the mechanisms underlying Wnt signaling is fundamental to studies of both normal and altered development, such as in wound healing and cancer, and has remained elemental in basic and applied biological research (3)(4)(5)(6)(7). There is, however, considerable debate about the spatial and temporal regulation and interaction of different Wnt signaling pathways; the distance and the mechanisms by which Wnts can travel across tissues; the mechanism by which distinct Wnt pathway members are regulated in cells; and how different Wnt ligands and receptors interact with each other (8)(9)(10).
In classical studies, there are two main pathways involved in Wnt signaling-a canonical and a noncanonical pathway-which use different ligand and receptor gene paralogs to transduce extracellular signals. In canonical Wnt signaling in Drosophila (fig. S1, A and B), specific Wnt ligands bind to specific Frizzled receptors at the cell surface, which then signal through Armadillo (Arm) (β-catenin in vertebrates) to regulate gene expression in the nucleus (11)(12)(13)(14). Noncanonical Wnt signaling works independently of Arm/βcatenin and includes the planar cell polarity (PCP) pathway, which regulates the cytoskeleton of cells ( fig. S1C), among other pathways (15)(16)(17)(18). PCP signaling has been reported to work both in a Wnt ligand-dependent and in a Wnt ligand-independent manner in mouse and Drosophila, respectively (15,19,20). Both canonical and noncanonical pathways, however, are involved in similar processes of tissue organization, cell proliferation, and cell-cell communication (2,9,(21)(22)(23).
The distinct Wnt ligands and Frizzled receptors used by Wnt pathways belong to multicopy gene families. In insect genomes, there are six to nine Wnts (24) and three to four frizzled genes (25), while in mammalian systems, there are 19 Wnts and 10 frizzled genes (9,18). Classically, Wnts such as Wnt1, Wnt3, Wnt8, and Wnt10 have been associated with the canonical pathway, while Wnt5a, Wnt7a, and Wnt11 with the noncanonical pathway in metazoans (9,(26)(27)(28)(29). Newer studies, however, have contradicted such categorization since some Wnts can transduce alternate Wnt signaling when coupled with specific receptors (9). Wnt5a, for example, can work both in the canonical pathway, using receptors such as frizzled4, and noncanonical pathway using frizzled8 receptors (28,30). Here, we examine the diversity of Wnt signaling pathways involved in butterfly wing patterning, as these large epithelial surfaces, with their diverse and colorful wing patterns, are ideal canvases for exploring complex pattern formation processes via cell signaling.
Wnt signaling, using Wnt1, has previously been implicated in regulating butterfly eyespot size, but the involvement of Wnt signaling in differentiating eyespot centers, critical for color ring differentiation, has not been investigated. Multiple butterfly species have stable Wnt1 expression along the wing margin throughout larval development (31)(32)(33)(34), but the signal transducer of canonical Wnt signaling, Arm, displays a dynamic pattern of expression during the larval stages (35,36). Arm starts to be expressed across the whole wing but later resolves into a broad vein and marginal expression. This expression then progressively narrows on top of the veins, wing margin, and also along fingers parallel to and centered between wing veins. These fingers contain an enlarged cluster of Arm-expressing cells in the middle, the eyespot foci, that mark the future eyespot centers (35,36). Later in development, during the early pupal stage, Wnt1 is expressed in these focal cells suggesting a role in signaling from these cells to pattern the rings of color around the focus (32,37). Transgenic RNA interference studies against Wnt1, where Wnt1 was down-regulated at the end of the pre-pupal stage and beginning of the pupal stage, resulted in the reduction of all the colored rings, indicating that Wnt1 regulates eyespot size (37). It is, however, still unclear whether Arm or Wnt signaling is required for eyespot center differentiation in the larval stages. Furthermore, the identity of the Wnt ligand(s) leading to Arm nuclear localization in the eyespot center during larval wing development is currently unknown.
WntA is another Wnt ligand that has been linked to the development and identity of cells involved in distinct color pattern elements including bands, patches, and eyespots in butterfly wings. Linkage mapping studies first identified WntA as an important prepatterning gene in Heliconius butterflies (38). Variation in WntA expression in different Nymphalid butterflies appears to correspond to the complex array of banding patterns laid out in the Nymphalid groundplan (39,40). In a generalized butterfly wing pattern, WntA is expressed in a central band [of the central symmetry system (CSS)], a more proximal band [of the basal symmetry system (BSS)], and along a marginal band system (MBS) in the larval wings of some Nymphalid butterflies including Junonia coenia, Pararge aegeria, Vanessa cardui, Agraulis vanilla, and Heliconius butterflies (34,38,41). WntA is also expressed in the silver color patches/spots in A. vanilla and in the ventral forewing eyespots of V. cardui (34,41). Knockout of WntA resulted in phenotypes with loss of pigmentation and reduction in the forewing eyespots of V. cardui (41). WntA has been shown to control the domain over which Optix is expressed, where knockout of WntA results in the expansion of Optix in Heliconius erato (42). In Bicyclus anynana, no study has yet explored the expression and function of this ligand.
In the present work, we explored the spatial and temporal expression of different Wnt signaling pathway genes in the wings of B. anynana, containing both eyespots and banding patterns. We first focused on the localization of all the Wnt ligands, frizzled receptors, Arm signal transducer, and the known Wnt target genes in Drosophila melanogaster, Distal-less (Dll) and vestigial (vg) in larval and pupal wings, and then we tested the function of four of these genes, arm, WntA, Wnt7, and frizzled4, with CRISPR-Cas9. Arm is present homogeneously throughout the larval forewing and hindwing tissue till stage 0.50. At stages 0.50 to 0.75, accumulation in the eyespot foci is observed that continues throughout the larval stage together with expression along the wing margin and the wing veins (shown here till stage 2.00); for stage nomenclature, please refer to (36). Scale bars, 250 μm. (C and D) During pupal forewing and hindwing development, Arm is observed in the foci from 15 to 24 hours after pupation (AP). (E and F) Arm is localized in the cytoplasm and in the nuclei of focal cells. (G) Outside the foci, Arm is present mostly at the cell membrane.

Phylogenetic analysis of Wnt and frizzled genes of B. anynana cluster with members of the same gene family
To discover all possible Wnt and frizzled genes present in the B. anynana genome, we searched for the corresponding gene annotations in National Center for Biotechnology Information [NCBI; B. anynana (taxid:110368)] and also blasted the orthologous Wnt and frizzled sequences from D. melanogaster in FlyBase against the B. anynana genome nBa.0.1, v1.2 and v2.0 in Lepbase (43)(44)(45). We identified eight Wnts and four frizzled genes in the genome. Wnt9 was only found in nBa.0.1 but not v1.2 and v2.0 of the B. anynana genome (46). A phylogenetic analysis performed with these genes and orthologs from other insects showed that the eight Wnts represent Wnt1, Wnt5, Wnt6, Wnt7, Wnt9, Wnt10, Wnt11, and WntA, who cluster with members of the same gene family from other butterfly and insect species (figs. S2 and S3). Similarly, a separate phylogenetic analysis identified frizzled, frizzled2, frizzled4, and frizzled9 as being the four frizzled genes in the B. anynana genome (figs. S4 and S5).

Arm is expressed in a dynamic pattern in larval and pupal wings
Previous gene expression/accumulation, transcriptomic, and modeling studies have proposed a role for Arm in the differentiation of the eyespot foci (35,36,47). To extend these findings, we used immunostainings to document Arm's spatial-temporal expression across both larval and pupal wings at multiple time points and across a longer period of wing development than previously investigated. In early fifth instar larvae, Arm was homogeneously distributed across the wing (stage 0.25; Fig. 1, A and B). As the wing developed, the protein was accumulated along the vein cells, wing margin, and at the eyespot foci (stages 0.50 to 2.00; Fig. 1, A and B). During the pupal stage, Arm protein was observed in the eyespot foci and along the wing margin from 15 to 24 hours after pupation (AP) (Fig. 1, C and D). We lack data from the early hours AP (before 15 hours AP) because wings are too fragile to handle before this stage. Arm protein was also observed in the foci in three other nymphalid butterflies during mid-late larval wing development along with the eyespot marker protein Spalt ( fig. S6). We conclude that Arm protein, and canonical Wnt signaling, is likely continuously present in the eyespot foci from mid fifth instar larval development to at least 24 hours AP. We observed cytoplasmic Arm in the eyespot center, while in the surrounding cells, Arm was present mostly in the cell membrane ( Fig. 1, E to G).
Knockout of arm using CRISPR-cas9 resulted in mosaic crispants that showed either a complete loss of the eyespot (one individ-   The mRNA expression of three canonical Wnts, Wnt1, Wnt6, and Wnt10, in the forewing and hindwing is limited to the wing margin and there is no focal expression. Expression is observed at the discal spot, for all three genes, during larval wing development (white arrows). Expression of (C and D) Wnt1 transcripts in the eyespot foci and in the wing margin (black/yellow arrows) and (E) Wnt6 and (F) Wnt10 in the wing margin (magenta and cyan arrow) from 18 to 21 hours of pupal wing development. Note the autofluorescence in the tracheal tissue running along the wing veins in some wings are not mRNA signals. stage, nuclear Arm at the foci is probably driven by Wnt1, Wnt6, and Wnt10 produced along the wing margin, some distance away, and reaching the focal cells via some form of diffusion (or other form of transport), as previously modeled (35). Expression of all the three Wnts was also observed in the discal spot, on top of a future cross-vein (Fig. 2, A and B, and fig. S8), consistent with previous studies in other butterflies (33,34).

Dll and vg are expressed in larval wings and Dll also in pupal wings
To investigate the domain over which Wnt1 glycoproteins, along with Wnt6 and Wnt10, might be activating target genes, we examined the coexpression of Wnt1 and two known targets of Wnt signaling in Drosophila, Dll and vg, in larval and pupal wings of B. anynana. In the Drosophila wing, disc Wnt1 is secreted from the wing margin and travels into more interior wing regions where it activates Dll and vg at two different concentration thresholds (1). In the early larval wings of butterflies, we observed the expression of Dll in broad finger-like projections some cells away from the wing margin (

Wnt5, Wnt7, Wnt9, and Wnt11 are not expressed in the stages tested during larval and pupal wing development
To test whether Wnt5, Wnt7, Wnt9, and Wnt11 typically associated with noncanonical Wnt signaling could play a role in B. anynana wing pattern formation, we also examined their expression in larval and pupal wings. None of the genes showed any specific expression domain in the larval and pupal wings at the stages tested ( Fig. 4 and fig. S12). However, knockouts of Wnt7 using CRISPR-Cas9 resulted in ectopic veins and ectopic eyespots differentiating in the wing sectors in 12 individuals (fig. S15, A to D). Because ectopic veins often lead to the creation of additional wing sectors with eyespots, we cannot directly implicate Wnt7 in eyespot development. This gene appears to play a role, however, in vein development.

WntA is involved in patterning the wings during larval and pupal wing development
We next tested the expression of the last Wnt ligand, WntA, in both the larval and pupal wings of B. anynana. In larval wings, WntA was  expressed along the CSS and along the MBS (Fig. 5, B and C, and fig.  S10A). In the pupal stage, strong expression of WntA was observed in two bands along the BSS and along the CSS in forewings and one band along the CSS in hindwings (Fig. 5, D to I, and fig. S10F). These results are consistent with studies in other butterfly species (41). No expression was observed in the eyespot region in larval and pupal wings (Fig. 5, B to I, and fig. S10). We used Optix, a known transcription factor in butterfly wing patterning (49), as a positive control spanning the boundary of WntA expression in the CSS (Fig. 5, D to I, and fig. S10, E to G).
Functional disruption of WntA using CRISPR-cas9 resulted in the loss of brown color with ectopic orange scales appearing along the CSS and in the BSS (Fig. 5, J to M) and in changes in the width and outer shape of the CSS in 28 individuals (Fig. 5, K and L). No defects in the eyespot pattern were obtained in any individual (Fig. 5, K to M), but the light-colored marginal chevrons of the wing were disrupted (compare left and right hindwings in Fig. 5K). We confirmed indels at the targeted sites using Illumina sequencing from the affected adult wings (Fig. 5N).

Expressions of frizzled4 and frizzled9 in larval wings are anti-colocalized
We next aimed to identify the Frizzled receptor(s) that might be used for Wnt signaling in larval wings. We first examined the expression of frizzled4 and frizzled9 transcripts in larval wings using HCR3.0. In early stages (0.75 to 1.0), frizzled4 was expressed homogeneously in the intervein cells (Fig. 6

Frizzled2 is expressed in a proximal domain while no specific domain of frizzled is observed in larval wings
We performed HCR3.0 on two other potential receptors, frizzled2 and frizzled, in both larval and pupal wings. In the larval stage, the expression of frizzled2 was restricted to the proximal domain of both fore and hindwings (Fig. 6, M to P). frizzled showed no specific expression in larval wings consistent with a previous study (50) (Fig. 6, Q and R, and fig. S13, Q to T) but was expressed in elevated levels in the eyespot foci in pupal wings at 18 to 24 hours AP (fig. S13, U to X).
frizzled2 is anti-colocalized with frizzled4 in the eyespot field, with frizzled9 in the pupal wing margin, and with WntA in the CSS band Next, we investigated the 18 to 24 hours pupal wing expression of frizzled2, frizzled4, and frizzled9. During the pupal stage, frizzled4 was expressed in the eyespot foci and in domains spanning the eyespot rings, bearing little resemblance to its early larval patterns (Fig. 7, A and B, and fig. S14C). frizzled2 was expressed in two large domains flanking each side of the CSS, where WntA was previously observed: a proximal band domain and a distal domain (Fig. 7C and figs. S10C and S14, A and B). In this distal domain, the expression of frizzled2 was elevated in the eyespot foci but reduced in the areas of the future black and orange rings (Fig. 7, C and D, and fig. S10C). Coexpression of frizzled2 and frizzled4 showed the eyespot domain over which frizzled4 was highly expressed had reduced levels of friz-zled2 (Fig. 7, E to G, and fig. S14, D to F). Lower levels of frizzled2 and frizzled4 were also observed along the wing margin, where friz-zled9 was strongly expressed (Fig. 7, D and H to K). The focal expression of frizzled4 overlapped the expression of Wnt1 (Fig. 7, L to N).

frizzled4 functions in eyespot center differentiation and in PCP
To test the role of frizzled4, we knocked it out using CRISPR-Cas9 at the embryonic stage. In 18 individuals, mosaic crispant wings differentiated two eyespot centers along the proximal-distal axis (Fig. 8, C to F and H, and fig. S15, E to K). Two crispants also showed defects in the orientation of the orange and black scale cells in eyespots (Fig. 8, H to J). The knockout results were confirmed by sequencing the affected wing tissue (Fig. 8K).

Wnt signaling is involved in eyespot center differentiation in B. anynana
Our results suggest that canonical Wnt signaling is involved in the differentiation of the eyespot centers during the larval stage. At this stage, the canonical Wnt signaling transducer Arm is accumulated at high levels in the fingers and in the nuclei of cells in the eyespot centers, indicating the activation of Wnt signaling in those cells (Fig. 1, E and F). Disruptions of arm via CRISPR-cas9 resulted in either a split eyespot or no eyespots, suggesting arm's essential role in eyespot center cell differentiation ( fig. S7, C to H). arm knockouts also led to disruptions in the wing margin, suggesting its involvement in margin differentiation ( fig. S7, C to L). The Wnt ligands Wnt1, Wnt6, and Wnt10, expressed along the wing margin throughout larval wing development (Fig. 2, A and B, and fig.  S8), are likely leading to arm cytoplasmatic accumulation along the margin, in the fingers that project from the margin, and in the eyespot focus.
We hypothesize that Frizzled9 is the receptor involved in signal transduction along the wing margin and in the eyespot center cells resulting in the accumulation of cytoplasmic Arm (Fig. 9A). For Wnt ligands to reach the focal cells some distance away from the wing margin, they need to have a Wnt receptor expressed in those cells, and Frizzled9 is likely that receptor (Figs. 6 and 10B and fig.  S13, I to P). This receptor is expressed not only along the wing margin but also in the intervein cells (Fig. 6, B and E, and fig.  S13, I and J) where Arm is also present (Fig. 1, A and B). In human cell culture experiments, Frizzled9 was shown to be involved in the activation of canonical Wnt signaling and in the accumulation of β-catenin (Arm) in the cytoplasm (51). While functional experiments are still required, we propose that during larval wing development, canonical Wnt proteins are secreted from the wing margin and captured by Frizzled9 in the wing margin, in the finger projections, and in eyespot foci. This results in the accumulation of cytoplasmic Arm in those cells and eventually nuclear Arm (Figs. 1, E and F, and 10B). In J. coenia and V. cardui, knockout of   The accumulation of Arm in the eyespot centers, however, may proceed through an interplay between canonical and noncanonical Wnt signaling (Fig. 9A). frizzled4, a receptor typically associated with noncanonical Wnt signaling (53) is anti-colocalized with Arm in the eyespot foci, finger projections, wing margin, and veins (Figs. 6A, E, H, and K, and 10A and fig. S13, A to H), all the areas where Arm is present (Fig. 1, A and B). The anti-colocalization of frizzled4 and Arm suggests that frizzled4 functions via a noncanonical mode. None of the proposed noncanonical Wnt ligands (Wnt5, Wnt7, Wnt9, and Wnt11) are coexpressed with friz-zled4 in the stages sampled (Figs. 4 and 10A and fig. S12), indicating that noncanonical Wnt signaling is likely transduced through friz-zled4 independently of any Wnt ligand, similar to what has been proposed for Drosophila wings (15,20). Disruptions of frizzled4 in B. anynana resulted in the appearance of two eyespots along the proximo-distal axis of a wing sector (Fig. 8, C to F, and fig. S12, E to K), not involving ectopic venation (fig. S15, H to K). These dual eyespots phenocopy a Dll overexpression phenotype previously studied and modeled via a reaction-diffusion mechanism using canonical Wnt signaling (35). We propose, thus, that friz-zled4-mediated signaling represses canonical Wnt signaling, as also observed in arterial network organization in mice (53). This repression would restrict canonical Wnt signaling (Arm accumulation) to regions of the fingers and eyespot focus. Removal of frizzled4 likely results in the up-regulation of arm and frizzled9, resulting in additional eyespot foci (Fig. 8, C to F).
We also propose that Arm accumulation in the eyespot centers during the larval stage leads to the activation of both Wnt1 (Fig. 2, C and D) and dpp [bone morphogenetic protein (BMP) signaling] (54) in the pupal wing eyespot centers. Activation of both Wnt1 and dpp signaling by Arm (β-catenin) has been shown in Drosophila, mice, and zebrafish in previous studies (55)(56)(57). Signaling by Wnt1 (and dpp) might be involved in setting up the rings of color around the center during the pupal stage (37).
Other frizzled receptors were either present in distinct patterns or absent in the larval wings in the stages tested. We did not detect  any expression of frizzled in the larval wing stages sampled, and friz-zled2 was expressed along a proximal wing domain. The presence of frizzled2 in the proximal domain is likely due to the repression of frizzled2 by Wnt signaling from the wing margin as previously shown in Drosophila larval wings (58).

The spatial regulation of frizzled2 likely involves repression from Wnt signaling at different domains of the pupal wings
During the first 18 to 24 hours of pupal wing development, we observed a very precise control of the domains over which the ligands Wnt1, Wnt6, and Wnt10; the receptors frizzled2, frizzled9, and friz-zled4; and the signal transducer Arm are expressed (Figs. 1, 2, and  7), suggesting that they are regulated (and cross-regulated) by the signaling pathways where they function. Both frizzled2 and friz-zled4, along with Arm, are expressed in the eyespot focal cells, but frizzled2 is anti-colocalized with frizzled4 in the rest of the eyespot field (Fig. 7, E to G, fig. S14, D to F). Cross-regulation might also be happening between a Wnt signaling pathway using Wnt1, Wnt6, Wnt10, frizzled9, and Arm, expressed along the wing margin (Figs. 1, C and D; 2, C to F; and 7H), and frizzled 2, expressed at lower levels in the margin and chevron area (Fig. 7C and fig. 14,  A and B). Similarly, WntA is anti-colocalized with frizzled2 along the CSS (Fig. 3 and fig. S10). The anti-colocalization of WntA with frizzled2 in the CSS is also consistent with expression and functional data in V. cardui where WntA and frizzled2 have anti-colocalized expression domains, and loss of WntA results in expansion of frizzled2 in the domains of WntA expressing cells (52). We propose a model (Fig. 9B) for the spatial regulation of friz-zled2 in B. anynana which involves down-regulation of this gene by Wnt signaling from the margin, from the eyespot centers, and along the CSS. frizzled2 might be initially homogeneously expressed in the pupal wing, but Wnt signaling from the wing margin (Wnt1, Friz-zled9, and Arm), the eyespot center (Wnt1, Frizzled4, and Arm), and along the CSS (WntA) represses the activity of frizzled2 in their respective domains resulting in the precise domains of friz-zled2 expression we observe in the pupal wings (Fig. 9B). This model is consistent with Wnt1-dependent signaling in Drosophila repressing frizzled2 in the dorsal-ventral axis of the wing disc and in the segments of the embryo (58,59).
frizzled4 is involved in regulating scale polarity in the eyespot field frizzled4 is also involved in the orientation of the scale cells in the eyespot field of B. anynana. The gene is expressed in the eyespot field during the pupal stage (Figs. 7, A and B, and 10A), and disruptions of frizzled4 resulted in scale orientation defects (Figs. 8, H to J, and 9C). The PCP pathway, however, is most probably independent of any Wnt ligand as previously shown in Drosophila wings (15,20). This pathway, when disrupted, results in changes in the orientation of cellular protrusions such as trichomes in Drosophila wings (15) and mouse skin (16). Scales appear to behave in the same way as trichomes in Drosophila wing, despite not being homologous traits. Butterfly wing scales are F-actin-based protrusions from the epithelial cells which are deposited with chitin and are homologous to bristles (60,61).
Wnt1 is likely functioning with Frizzled2, Frizzled4, and Arm in pupal wings to determine eyespot size Wnt1 was previously proposed as a morphogen produced in the eyespot centers (32) to regulate eyespot size in B. anynana (37). The mechanism by which Wnt1 operates in the determination of the eyespot rings, however, has not yet been explored. Here, we confirmed the presence of Wnt1 mRNA in the eyespot central cells during early pupal wing development coexpressed with frizzled2, frizzled4, and Arm (Fig. 10A) (37). The Wnt1 ligand is likely being secreted by the central cells and captured by the receptors Frizzled4 and Frizzled2 (to a lesser extent), whose transcripts are expressed across the whole eyespot field (Figs. 7, A to D, and 10C; and fig. S14, A to C). Active canonical Wnt signaling via the Frizzled receptors should result in higher levels of Arm in the eyespot rings. We, however, only observed strong Arm accumulation in the center of the eyespot during pupal wing development (Fig. 1, C and D). Elevated cytoplasmic Arm levels in the eyespot central cells likely happen due to the coexpression of frizzled2, frizzled4, and frizzled (Fig. 7, A to D, and figs. S13, U to X, and S14) during pupal wing development. In the rest of the eyespot field, Wnt signaling is probably being transduced via canonical signaling with lower levels of Arm which are difficult to detect via immunostaining.

WntA appears to function as a morphogen and as a scale color regulator at different stages of wing development
We observed two distinct phenotypes in B. anynana WntA crispants that suggest that this gene may have two distinct functions. The most obvious phenotype was the loss of brown scales in the CSS and BSS bands and the appearance of orange scales in their place. The appearance of orange scales is likely due to the ectopic expression of Optix in those regions, a gene that might normally be repressed by WntA in the pupal stage (Fig. 5, K to M). Such ectopic expression of Optix was observed in H. erato pupal wings in WntA crispants (42). The less obvious phenotypes were the narrower and distorted bands of the CSS and the loss of the light chevron pattern marginal elements (Fig. 5K). These two effects suggest that WntA might also function as a morphogen to define the width of the CSS and the MBS. This function may take place during the larval stage, as WntA is expressed in the wing margin only at this stage and is expressed in a narrower domain in the center of the future CSS band (Fig. 5, B and C). WntA likely operates noncanonically, independently of Arm since no Arm expression is observed in the domains over which WntA has strong expression, except for the wing margin ( Figs. 1 and 3, B to I). This noncanonical signaling of WntA is consistent with a similar hypothesis in other butterflies (52). WntA has also been shown to be involved in the patterning of the BSS, the CSS, and the MBS in J. coenia, Pararge aegeria, V. cardui, A. vanilla, and Heliconius butterflies (34,38,41) and in the eyespots of V. cardui (41,52). More recently, WntA has also been proposed as a morphogen that induces cell differentiation in a concentration-dependent manner at some distance away from producing cells (52).
We note that the discal spots are not affected in WntA knockout adults (Fig. 5, K to M). Other Wnts (Wnt1, Wnt6, and Wnt10) are expressed in this domain and are likely important for the identity of the cells there (Fig. 2, A and B, and fig. S8). Future studies with functional knockouts of these ligands will unravel their role in the discal spot. The expression of Wnt1, Wnt6, and Wnt10 along in this spot is conserved across butterflies and likely plays a similar role in discal spot development (34).
In summary, we illustrated the expression and function of different Wnt signaling pathway members in larval and pupal wings of B. anynana wings that contribute to the precise differentiation of eyespots, bands, and chevron patterns. Further studies are necessary to examine the function of several of these genes, especially that of friz-zled9, and the interaction between frizzled4, frizzled9, and frizzled2. The interaction of Wnt signaling with BMP also needs further study, as this has been proposed to function in the reaction diffusion mechanism for eyespot center formation during the larval stage (35) and ring differentiation during the pupal stage (54). The Wnt signaling mechanism for eyespots, however, is likely conserved in other nymphalid butterfly species, as observed with the expression pattern of Arm ( fig. S6) and canonical Wnt ligands (34). Studying complex signaling pathways, such as Wnt signaling, in simple twodimensional (2D) surfaces such as butterfly wings may help unravel the function of this pathway in more complex 3D traits such as legs (62,63), antennae (45), and horns (64,65), where Wnt signaling is also fundamental to trait development and toward our understanding of this signaling in higher animals.

Sequence alignment and phylogenetic tree construction
Nucleotide sequences were obtained from NCBI and FlyBase. Alignments were carried out using ClustalW (66) with the default parameters in "SLOW/ACCURATE" alignment option in GenomeNet.
Using RAxML (Randomly Accelerated Maximum Likelihood) v8.1.20 a maximum likelihood tree was created with model PROT-GAMMAJTT and default parameters with 100 bootstraps (67)  No animal experimentation permits were required for the experiments conducted here. B. anynana butterflies have been reared in the lab since 1988 and imported under an Agri-Food & Veterinary Authority permit to the lab in Singapore.

CRISPR-Cas9
arm, WntA, Wnt7, and fz4 embryonic CRISPRs were carried out on the basis of the protocol described in (45,70) (table S2). One to two RNA guides were designed using CRISPRdirect or CHOPCHOP to target the coding sequence of these genes (see the Supplementary Materials for sequences). Cas9 protein (Integrated DNA Technologies, catalog no. 1081058) (300 ng/μl) along with the guide RNA at a concentration of 300 ng/μl were mixed in molecular grade water with Cas9 buffer (New England Biolabs, catalog no. M0386S). Embryos were injected 6 hours after egg laying for arm, 1 to 3 hours for WntA, and 3 hours for frizzled4 and Wnt7. Embryos were kept in petri dishes inside a temperature-controlled incubator with moistened cotton to maintain humidity. The larvae were reared in mesh cages and fed young corn leaves. After pupation, the larvae were transferred to plastic containers, and after eclosion, the adults were frozen at −20°C and imaged under a Leica DMS1000 microscope. Scales were bleached using sodium hypochlorite (Clorox).
For next-gen sequencing, DNA was extracted from affected wings using an Omega tissue DNA extraction kit (catalog no. D3396-01). For the arm and WntA crispants, primers flanking around the CRISPR target site were used to amplify the region of interest (primer sequences in table S1). For arm, adapters and indices were added to the polymerase chain reaction (PCR) product via a two-step PCR reaction (table S1), followed by purification of the PCR products. The samples were sequenced using an Illumina iSeq 100 sequencer. For WntA, the amplicon was sent to Azenta Inc. for Illumina sequencing. Reads were aligned to the reference wild-type (WT) arm and WntA sequences using Geneious R10 (71).
For Sanger sequencing (fz4) of the crispants and WT, DNA was extracted as described above using an Omega tissue DNA extraction kit. PCR was amplified using the gene-specific primers (primer sequences in table S1) and purified. Sequencing was carried out at 1st BASE, Singapore. CRISPR indel was analyzed using Synthego Performance Analysis, ICE Analysis. 2019. v3.0. (last accessed October 2022) (https://synthego.com/products/bioinformatics/crispranalysis).

Immunostainings
The moment of pupation was timed using an Olympus tough TG-6 camera, and pupal wings were dissected and stained on the basis of a protocol described in (36). Briefly, wings were dissected in 1× phosphate-buffered saline (PBS) under ice for larval wings and in 1× PBS at room temperature for pupal wings. Afterward, wings were transferred to fix buffer (table S3) with 4% formaldehyde in ice for larval wings and at room temperature for pupal wings. After fixation, the wings were transferred to ice, washed with 1× PBS, and kept in block buffer (table S3) (table S3). Last, the wings were mounted in an in-house mounting media (see table S3) and imaged under an Olympus fv3000 microscope.

Chromogenic based in situ hybridization
In situ hybridization experiments were carried out on the basis of the protocol described in (36). Pupal wings were dissected in 1× PBS and fixed in 4% formaldehyde in 1× Phosphate Buffer Saline with Tween20 (PBST) at room temperature. After fixation, wings were treated with proteinase k for 5 min and afterward with glycine (100 mg/ml) in 1× PBST. Wings were then washed with 1× PBST and transferred to prehybridization buffer (table S4) at 65°C followed by incubation in hybridization buffer (table S4) with probes against Wnt1 (see the Supplementary Materials for sequences) at 65°C for 16 to 24 hours. After hybridization, wings were washed with prehybridization buffer at 65°C, five times, for 30 min each time. Afterward, wings were brought down to room temperature and washed four times with 1× PBST. Wings were then incubated in block buffer (table S4) for 60 min, followed by incubation in anti-digoxygenin (Sigma-Aldrich, catalog no. 11093274910) at the concentration of 1:2000 in block buffer. The wings were then washed five times with a block buffer, 10 min each time. The wings were then washed two times in alkaline-phosphatase buffer, for 5 min each time. After washing, wings were transferred to alkaline-phosphatase buffer supplemented with bromochloroindolyl phosphate-nitro blue tetrazolium (Promega, catalog no. S3771) and left for 4 to 6 hours in the dark for color to develop. Once the color developed, wings were washed two times with 1× PBST and mounted in 60% glycerol, and imaged under a Leica DMS1000 microscope.
Fluorescent based in situ hybridization (HCR3.0) Fluorescent in situ hybridization was performed on the basis of the protocol developed by Choi et al. (48) with a few modifications optimized for butterfly wing tissue. Briefly, wings were dissected in 1× PBS and fixed in 4% formaldehyde in 1× PBST. After fixation, wings were washed with 1× PBST and permeabilized using a detergent solution (73). The wings were again washed with 1× PBST and with 5× Saline-Sodium Citrate with Tween20 (SSCT) followed by the addition of 30% probe hybridization buffer. Afterward, wings were incubated overnight at 37°C in a chamber with 30% probe hybridization buffer and HCR3.0 probes. The next day, wings were washed five times with 30% probe wash buffer at 37°C followed by two washes with 5× SSCT at room temperature. Wings were then incubated in an amplification buffer with secondary fluorescent hairpin probes (Molecular Instruments) in the dark for 16 to 20 hours. The next day, wings were washed four times in 5× SSCT, mounted in an in-house mounting media, and imaged under an Olympus fv3000 microscope.